Abstract

Hereditary cancer syndromes providepowerful insights into common forms of cancer. They lead to further understanding of the somatic mutations presentin sporadic cancers, as well as the function of cell signalingpathways. The \#946;-Spectrin and adaptor protein ELF (Embryonic Liver Fodrin) is a potent regulator of tumorigenesis through its ability to affect TGF-\#946; tumor suppressor function, specifically Smad3 and Smad4 signaling. We show that elf+/- and elf+/-/Smad3+/-mice (C57 B6) spontaneously develop multiple cancers and a nearly identical phenotype observed in patients with Beckwith-Wiedemann syndrome (BWS). BWS is a hereditary cancer overgrowth syndrome and epigenetic disorder that remains poorly understood in the majority of cases, and is associated with an 800-fold increased risk of embryonal neoplasms of childhood that include Wilms\#8217; tumors, hepatoblastomas, pancreatoblastoma, neuroblastoma, rhabdomyosarcoma, and adrenocortical carcinomas. Results show that: 1) anelf+/- and elf+/-/Smad3+/-mice develop the abnormal ear folds, visceromegaly, adrenal cytomegaly and multiple cancers including liver and metastatic pancreatic cancers observed in patients with BWS. 2) Loss of ELF, but not Smad3 or p53 RNA is observed in elf+/-, elf+/-/Smad3+/- tumors, BWS tissues, BWS cancers and cell lines. 3) Exogenous ELF rescues TGF-\#946; signaling correcting aberrant Smad3 localization, and TGF-\#946; target gene activation in BWS cell lines. 4) ELF-TGF-\#946; signaling is required for suppression of IGF2 signaling, suggesting that the increased expression of IGF2 observed in BWS may be secondary to loss of ELF, and not the causal event in BWS. 5) In 6 of 7 human BWS cell lines and liver, kidney and tongue tissue, MS PCR and bisulphite sequencing consistently demonstrate loss of ELF through DNA methylation of the ELF promoter irrespective of IGF2 LOI status; 6) 5\#8242;-aza-2\#8242;-deoxycytidine, an inhibitor of DNA methylation, reactivates Elf gene expression in BWS cell lines. Conclusions: Our results suggest that epigenetic regulation of the TGF-\#946; pathway results in the loss of ELF and its crucial Smad3 adaptor function. The loss of the \#946;-Spectrin, non-PH domain\#8217;s spectrin ELF, is causally related to the genesis and progression of human BWS. In addition, elf+/- and elf+/-/Smad3+/-mice provide an important animal model for BWS, as well as to give crucial insight into sporadic lethal cancers such as hepatocellular and metastatic pancreatic cancers observed in the mice and BWS patients.